Diagnosis of collagen IX destruction

ABSTRACT

A method for detecting or monitoring the presence of protein fragments, cleaved at novel cleaving sites near the N-terminal part of the collagen IX alpha 1 chain, close to the C-terminal part of the NC4 domain, and at the COL3 domain close to the NC3 domain. Neoepitope antibodies against the neoepitopes were created by the cleavages and an epitope in the cleaved N-terminal part of the NC4 domain unique to collagen IX. A diagnostic kit and antibodies useful in carrying out such methods are also presented.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 12/072,428, filed Feb. 26, 2008, now U.S. Pat. No. 7,892,768,which claims priority from U.S. provisional application Ser. No.60/904,684 filed Mar. 2, 2007.

FIELD OF THE INVENTION

The present invention relates to methods directed to detecting ormonitoring disorders affecting connective tissues, such as joint diseaseparticularly, osteoarthritis, rheumatoid arthritis, reactive arthritis,tendon rupture, osteoporosis, osteomalacia, fracture repair andarteriosclerosis, by determining the levels of cleaved fragments ofcollagen IX, mediated by IL-1, in serum and synovial fluids. Morespecifically, the present invention is related to detecting ormonitoring the presence of protein fragments, cleaved at novel cleavingsites near the N-terminal part of the collagen IX alpha 1 chain or morespecifically close to the C-terminal part of the NC4 domain, and at theCOL3 domain close to the NC3 domain. The invention also disclosesneoepitope antibodies against the neoepitopes created by the cleavagesand an epitope in the cleaved N-terminal part of the NC4 domain uniqueto collagen IX. A diagnostic kit and antibodies useful in carrying outsuch methods are also presented.

BACKGROUND

In hyaline cartilage type IX collagen there is a minor constituent ofthe fiber network having type II collagen as a major constituent. Thetype IX collagen molecule is a heterotrimer consisting of polypeptidechains α1, α2, and α3 (2). It belongs to the fibril-associated collagenswith interrupted triple-helix (FACIT). Each chain contain threetriple-helical (collagenous) domains COL 1, 2, and 3 surrounded by fournon-triple-helical domains, NC 1, 2, 3, and 4 (3), (see also FIG. 9).The domain numbers are counted from the C-terminal.

Using electron microscopy, it has been shown that type IX collagendecorates the surface of type II collagen fibrils and that the NC 4domain forms a globular structure, which together with the stalk likeCOL 3 domain protrudes out from the type II collagen fibril (4).

Type IX collagen is covalently crosslinked to the type II collagenfibrils through binding to both type II collagen and other type IXcollagen molecules (5-7). These bonds render extraction of type IXcollagen from mature cartilage virtually impossible by agents that donot cleave peptide bonds.

The NC 4 domain has been shown to have an affinity for a number ofmolecules, for example heparin and cartilage oligomeric matrix protein(COMP) (8-10), whereas COL domains interacts with matrilin-3 (11).Mutations in the interactive COMP, MATN-3, and COL9 have been found inpatients with multiple epiphyseal dysplasia, a rare inheritedheterogeneous range of diseases, affecting growth and ossification ofthe epiphysis (12).

NC4-fragments have now surprisingly been shown to be biomarkers forcartilage destruction. The cleavage occurs after aggrecan, a majorstructural component of cartilage is released, and prior to the majorcollagen degradation. NC4-fragments are markers of the process leadingto destruction before the “point of no return” in contrast tocollagen-markers that are like x-rays observations a result of the latestages of the destruction process. Aggrecan-related markers are releasedin an early stage of the degradation progress and also during normaladaptation of the tissue and therefore do not specifically represent apathological process. Furthermore aggrecan markers are often associatedto inflammation.

After the cleavage of NC4, described in this invention, the large partof type IX collagen that remains crosslinked to type II collagen ispotentially further cleaved and released at a later stage. Theneoepitope antibody disclosed in the invention herein, against the newC-terminal of the released fragment could detect such cleavage.Additionally, neoepitope antiserum disclosed in the invention, could beused in immunohistochemistry to determine were in the cartilage(superficial—deep, pericellular—interterritorial) the novel cleavagereported here has occurred. An antibody to the newly formed N-terminalof the fragment initially remaining in the tissue could detect a laterstage release of such fragments. Additional information on timing couldbe obtained using the neoepitopes created by cleavage in the col3domain. A number of earlier publications and patent-applicationsdescribe monoclonal antibodies that bind specifically to Type IXcollagen, but none of them mentions neoepitope antibodies specificallyagainst cleavage sites near NC4.

Patent application WO1990008195 A1 discloses oligopeptides correspondingto segments of human Type IX collagen useful as antigens in producingmonoclonal antibodies which bind immunologically to human Type IXcollagen and fragments derived from it. However the segments mentionedin the patent application are not involved in pathological destructionof collagen IX, and therefore not expected in connection to arthritis.

In WO2004110475 A1, assigned, Inst of Nutraceutical Research PTY, theinvention relates to compositions comprising the NC4 domain, fortreatment and tolerance induction to individuals with symptoms ofarthritis. The inventors are also describing methods for recoveringpolypeptides from connective tissue, having anti-arthritic oranti-inflammatory activity. As in the above-mentioned patent-applicationthe antibody is against uncleaved Collagen IX and not a neoepitopeantibody. Furthermore the applicants do not mention NC4 as a marker fordiagnosis.

The patent-application WO1999021011 A1, assigned Fibrogen Inc, reveals amethod for detecting or monitoring autoimmune disorders and connectivetissue disorders by determining the levels of type IX collagen in serum.When preparing antibodies against type IX collagen a sequence in theN-terminal portion of the NC4 domain was selected. This antibody isagainst uncleaved Collagen IX and not to unique cleavage neoepitopeslike in the invention herein.

None of the previous documents describes a specific cleavage siteoccurring in the NC-4 domain leading to exclusive release of thisdomain. Although the fact that tissues are completely destroyed incertain diseases, the character of the cleavage cannot be inferred orpredicted. It was also unexpected that the cleavage of the molecule inbovine cartilage and human cartilage is different, particularly in viewof the sequence similarities (FIG. 10).

Thus, the discovery of specific cleavage sites is novel and opens up forthe new diagnostic inventions.

Therefore we have developed a sensitive Western blot technique and asensitive inhibition ELISA as methods for the identified novel cleavagesite facilitating analysis of collagen IX destruction in any humansample, such as synovial fluid, serum, urine and any body fluids. Thiskind of assay could be used to establish if and when the identifiedcleavage appear in cartilage degradation and joint deterioration inpatients. By using the specific neoepitope antibody of the inventionherein, the assay can be used for diagnosis of disease progression andmonitoring disease response of treatment. For example to determine theoptimal moment for a sportsman with a healing bone fracture to return tothe training program.

SUMMARY OF THE INVENTION

Interleukin-1 induced bovine nasal cartilage degradation was used tostudy catabolic events in the tissue over a 16-day period. Culturemedium was fractionated by 2D-electrophoresis (isoelectric focusing andSDS PAGE). Identification of components by peptide mass fingerprintingrevealed release of protein fragments such as the NC4 domain of the typeIX collagen α1 chain at days 12 and 16.

A novel peptide antibody against an epitope in the N-terminal part ofthe NC4 domain confirmed the finding and indicated the presence of oneof the fragments already at day 9.

Mass spectrometric analysis of the two most abundant fragments revealedthat the smallest fragment contained almost the entire NC4 domain,cleaved between arginine-258 and isoleucine-259 in the sequence gives aC-terminal end: ETCNELPAR- (SEQ ID NO: 1) and a N-terminal end:-ITPGARSP (SEQ ID NO: 2) (ETCNELPAR²⁵⁸-COOH and NH₂-ITPGARSP).

A larger fragment contained the NC4 domain and the COL3 domain with acleavage site located between glycine-400 and threonine-401 in COL3gives a C-terminal end: RGPPGPPGPPGPSG- (SEQ ID NO: 3) and a N-terminalend: -TIGFHDGD (SEQ ID NO:4) (RGPPGPPGPPGPSG⁴⁰⁰-COOH and —NH₂-TIGFHDGD)close to the NC3 domain. Presence of multiple collagen α1 (IX)N-terminal sequences demonstrate that the released molecules are cleavedat sites very close to the original N-terminal, either prior to or dueto IL-1 treatment.

It has been shown (1) that matrix metalloproteinase 13 (MMP-13) isactive and cleaves fibromodulin in this time interval. Cartilageexplants treated with MMP-13 were shown to release collagen α1 (IX)fragments with the same sizes and with the same cleavage sites as thoseobtained upon IL-1 treatment. The presented data describe a potentiallyimportant degradation event apparently involving MMP-13 and thatprecedes the final type II collagen loss.

Further studies of cleavage of collagen IX in human cartilage, revealedthat there was specific cleavage sites also of the human collagen IX α1chain in the NC-4 domain but this surprisingly occurred at a sitedifferent from that of the bovine collagen. Cleavage in the site betweenglutamine-263 and threonine-264 in the CHELPARITPSQTTDERGPP (SEQ IDNO:5); of the human collagen IX alpha 1 chain in the NC-4 domain gives aC-terminal end: CHELPARITPSQ- (SEQ ID NO: 6) and a N-terminal end:-TTDERGPP) (SEQ ID NO: 7). (-CHELPARITPSQ²⁶³—COOH and NH₂-TTDERGPP). Thelarger fragment, representing the site in the COL3 domain, is of thesame size derived from bovine and human cartilage and cleavage occur atthe same site between G400 and T401 despite that the S399 in the bovineis exchanged for R399 in the human RGPPGPPGPPGPRG⁴⁰⁰ TIGFHDGD⁴⁰⁸ (SEQ IDNO:8). The cleavage of the human RGPPGPPGPPGPRG⁴⁰⁰ TIGFHDGD⁴⁰⁸ (SEQ IDNO:8), results in two neoepitopes RGPPGPPGPPGPRG (SEQ ID NO:9) andTIGFHDGD⁴⁰⁸ (SEQ ID NO:10). The antibodies to all the new neo-epitopeends of the human NC-4 and COL3 ware developed, using standard methods,and showed specific reactivity with the cleavage fragment. Theantibodies were used to develop ELISAs for the detection of theepitopes. These ELISAs were used to show presence of the epitopes insynovial fluid from patients with disorders affecting connectivetissues, such as joint disease particularly osteoarthritis andrheumatoid arthritis, tendon rupture, fracture repair andarteriosclerosis. The features of the present invention will be moreclearly understood by reference to the following examples, which are notto be construed as limiting the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Shows proteins from IL-1 stimulated bovine nasal cartilagemedium day 16 separated using 2D SDS PAGE. The first dimension,isoelectric focusing, was over a pH range 3-10, and the second dimensionutilized a 4-20% gradient gel. The numbers to the left of the picturerepresents the positions of reference marker proteins. Spots marked withrings show those that have been identified as collagen α1 (IX). Thedifferent type IX collagen fragments are in the text referred to as the28 kDa fragment, the 50 kDa fragment, and 100 kDa. In the pictureexamples of specific spots analyzed have been marked 5, 8, and 14. Theresults from MALDI-TOF MS (Matrix Assisted Laser Desorption IonizationTime Of Flight Mass Spectrometry) of these spots are presented in theFIGS. 11 a, b, and c below.

FIG. 2. Shows western blot with the NC4-antiserum of IL-1 stimulatedcartilage explants. Medium from day 6, 9, 12, and 16 of IL-1 stimulatedcartilage cultures, were separated on 1D SDS PAGE gel. A positive signalat apparent mass of 28 kDa, 40 kDa, and 50 kDa are clearly visible.

FIG. 3. Shows tandem mass spectrometric data from the most C-terminalpeptide of the 28 kDa NC4 fragment. The 28 kDa fragment was in-geldigested with AspN and analyzed with reversed phase LC (liquidchromatography, C18 column) on line with ESI-Qtof MS (ElectroSprayIonization-Quadrople time of flight mass spectrometry). The deducedpeptide sequence is ETCNELPAR (SEQ ID NO:1). The medium from IL-1stimulated cartilage (day 16) had first been separated on 2D SDS PAGE.This annotated spectrum was produced in the BioLynx software (Peptidesequencing, Waters Djupdalsvägen 12-14 Sollentuna Sweden). The peptidedelta mass is <0.01 Da, the average error for y-ions 0.01 Da, and theintensity threshold was set to 0.75%.

FIG. 4 a. Shows western blot of media from IL-1 and MMP-13 treatedcartilage using the NC4-antiserum. Proteins released from bovine nasalcartilage treated with IL-1 (day 16) or MMP-13 were separated by 16%-SDSPAGE. The equivalent positions on a Coomassie blue G-250 stained gelwere excised, in-gel digested and analyzed with by reversed phasechromatography (C18 column) on line with ESI-IonTrap MS (ElectroSprayIonization Ion Trap mass spectrometry). Both 28 kDa fragments containedthe same most C-terminal peptide ETCNELPAR (SEQ ID NO:1) (see FIG. 3).The 50 kDa fragments shared a common most C-terminal peptideRGPP*GPP*GPP*GPSG (SEQ ID NO:3) where P* denotes hydroxyprolines (seeFIG. 4 b). The insert to the right is an overexposed version of the sameblot illustrating the 40 kDa band.

FIG. 4 b. Shows tandem mass spectrometry of the most C-terminal peptidein the 50 kDa fragment of bovine collagen IX. Tandem mass spectrometricdata from the most C-terminal peptide (RGPP*GPP*GPP*GPSG (SEQ ID NO:3),where P* denotes Hydroxyproline residues) found when the 50 kDa fragmentwas in-gel digested with trypsin and analyzed with reversed phasechromatography on-line with IonTrap MSMS. The MMP-13 digest solution hadbeen separated on 16%-SDS PAGE and stained with Coomassie blue G-250.This annotated spectrum was produced in BioTools (Bruker DaltonicsScandinavia AB, Polygonvägen 79 Täby Sweden). The tolerance error was0.2 Da, average error for b- and y-ions were 0.06 Da, and the intensitythreshold was set to 10.

FIG. 5 a. Shows western blot, with the NC4 antiserum, of MMP-13 or IL-1treated human cartilage. Human articular and bovine nasal cartilageswere digested with MMP-13 and the latter was also stimulated with IL-1(day 16). The digest solutions and the medium were separated on a 16%polyacrylamide-SDS gel.

FIG. 5 b. Shows tandem mass spectrometric data from the most C-terminalpeptide of the human NC4 fragment. The fragment was in-gel digested withAsp-N and analyzed with LC-Qtof MS. Leucine and isoleucine have theexact same mass, thus L in the spectrum represents either L or I. Thededuced peptide sequence is ETCHELPARITPSQ (SEQ ID NO:11). The mediumfrom cartilage digested with MMP-13 had been fractionated on a 16%polyacrylamide-SDS gel prior to mass spectrometry.

FIG. 6. Shows western blot, using the neoepitope antiserum, of themedium from MMP-13 digested human articular cartilage. Human articularcartilages were digested with MMP-13. The digest solution was separatedon a 16% polyacrylamide-SDS gel and electrophoretically transferred to anitrocellulose membrane, which was incubated with a rabbit antiserumagainst an internal epitope in NC4 recognizing all variants (rightpanel) and the neoepitope antibody to PARITPSQ²⁶³ (SEQ ID NO:13), leftpanel. Bound antibodies were detected with a peroxidase conjugatedanti-rabbit IgG antibodies. The absence of neoepitope reactivity withthe larger fragment containing the continuous sequence over the cleavagesite demonstrates specificity for the cleaved fragment.

FIG. 7. Shows tandem mass spectrometric data (LC-ESI-QT of MS) of thepeptide SNSDGENELCPK (SEQ ID NO:12) in the NC4 domain, that exhibits anon-tryptic N-terminal cleavage. Bands were excised from the gel anddigested with trypsin followed by reversed phase chromatography withon-line ESI-Qtof MS. The peptide is deamidated, such that the D(aspartate) in this peptide sequence is an N (asparagine) in the bovinesequence gi|119901059). This annotated spectrum was produced in BioLynx(Peptide sequencing, Waters Djupdalsvägen 12-14 Sollentuna Sweden). Thepeptide delta mass is 0.01 Da, the average error for y-ions <0.02 Da,and the intensity threshold was set to 2%. From the same experiment asdata presented in FIG. 12 c.

FIG. 8. A) Is a graph showing immunoassay using the neoepitopeantiserum, comparing RA and OA samples with and without SDS. Synovialfluid from one RA and one OA patient were analyzed with or without SDS.The samples were diluted 1:10, 1:40, and 1:160.

B) Is a graph showing Western blot of synovial fluid samples frompatients with joint disease by SDS-polyacrylamide electrophoresis on4-16% gels followed by electrophoretic transfer to a nitrocellulosemembrane add staining with the antibody raised against the PARITPSQ (SEQID NO:13) cleavage neoepitope. Bound IgG was detected using a peroxidaseconjugated anti-rabbit IgG antibody. Note the presence of a 20 kDafragment in select synovial fluids containing the neoepitope. Thefragment is somewhat smaller than the one released from articularcartilage upon MMP-13 digestion.

FIG. 9. Is a schematic picture of the type IX collagen molecule withindicated cleavage sites. The thin line represents the non-collagenousdomains (NC) and the thick line represents the collagenous domains(COL). The arrows indicate the position of cleavage sites identified anddescribed.

FIG. 10. Cleavage sequences of collagen IX in human and bovinecartilage, respectively.

FIG. 11 a. Is a table showing the MALDI MS analysis result of spot 5from the 2D gel in FIG. 1. Database searches using the Mascot PeptideMass Fingerprint software (Matrix Science Inc, Boston, Mass., USA)identified collagen α1 (IX). Only peptides from the NC4 domain werematched to peaks in the spectrum. The matched peptides are tabulatedwith start and end position relating to the bovine sequence:gi|119901059. M_((expt)) is the measured monoisotopic mass of thepeptide, H is a hydrogen, “Delta” means the difference between themeasured and the theoretically calculated mass of a matching peptide.“Miss” shows how many missed cleavages that are present in a matchedpeptide sequence. “Sequence” shows the sequence of the matching peptidewith one extra amino acid on both the N- and the C-terminal separatedwith a hyphen. “Modifications” describe modifications in a peptidesequence, required to match it to an M_((expt)) obtained with massspectrometry. Allowed modifications (variable) were oxidation ofmethionines and hydroxylation of proline residues to formhydroxyproline. All cysteine residues were thought to becarbamidomethylated hence they were not listed.

FIG. 11 b. Is a table showing the MALDI MS analysis results of spot 8from the 2D gel in FIG. 1. Subsequent database search using the MascotPeptide Mass Fingerprint software identified collagen α1 (IX). Peptidesin both the NC4 and the COL3 domain matched to peaks in the spectrum.After the analysis of the 50 kDa fragment released from MMP-13 treatedcartilage explants (FIG. 4), this spectrum was inspected for thepresence of a peak matching the cleavage site peptide. It was alsopresent in these MALDI data (Observed mass=1274.628). The matchedpeptides are tabulated with start and end position relating to thebovine sequence: gi|119901059. See description in FIG. 11 a for tableexplanation.

FIG. 11 c. Is a table showing the MALDI MS analysis results of spot 14from the 2D gel in FIG. 1. Subsequent database search using the MascotPeptide Mass Fingerprint software identified collagen α1 (IX). Peptidesfrom NC4, COL3, and COL2 domains matched to peaks in the spectrum. Thematched peptides are tabulated with start and end position relating tothe bovine sequence: gi|119901059.

See description in FIG. 11 a for table explanation.

FIG. 12 a. Is a table showing peptides matching bovine collagen α1 (IX)according to a database search using Mascot “MSMS Ions search” (MatrixScience Inc. Boston, Mass., USA). The 28 kDa fragment was cut out from a2D gel, in-gel digested with AspN, and analyzed using the reversed phasenano-LC on-line with ESI-Qtof MS/MS. This represents data from the sameexperiment as shown FIG. 3. “M(expt)” means the monoisotopic mass of thematching peptide, otherwise the notations are the same as in FIG. 11.Ion score is based on the probability that an observed match is a randomhit (41). Higher values indicate a better hit. The matched peptides aretabulated with start and end position relating to the bovine sequence:gi|119901059.

FIG. 12 b. Is a table showing absence of putative trypsin generatedsites in the AspN digest of the 28 kDa bovine fragment. The sameexperiment as in FIG. 3 and FIG. 12 a, but searching for peptides in theAspN digest matching putative “tryptic” peptides reveals the cleavagesite peptide. No other peptides typical for trypsin digestion wereidentified. The matched peptides are tabulated with start and endposition relating to the bovine sequence: gi|119901059.

FIG. 12 c. Is a table showing peptides matching bovine collagen α1 (IX)in the 28 kDa band. The 28 kDa band was cut out from a 2D gel, in-geldigested with trypsin, and analyzed using the Qtof setup and data wereused to search the NCBI nr database with Mascot “MSMS Ions search”. Thematched peptides are tabulated with start and end position relating tothe bovine sequence: gi|119901059 (FIG. 7). “Modifications” describemodifications in a peptide sequence, required to match it to an m/zobtained with mass spectrometry. Allowed modifications (variable) wereoxidation of methionines, deamidation (asparagine and glutamine) andhydroxyprolines. All cysteine residues were thought to becarbamidomethylated, hence they were not listed.

FIG. 13. Is a table showing peptides matching the bovine collagen α1(IX) chain. The 50 kDa MMP-13 generated sample was cut out from a 1Dgel, in-gel digested with trypsin, and analyzed using the reversed phaseLC ESI-IonTrap MS setup. Data was further analyzed by a database searchusing Mascot “MSMS Ions search”. This was data from the same experimentas FIG. 4 b. The matched peptides are tabulated with start and endposition relating to the bovine sequence: gi|119901059. See FIGS. 12 aand 12 c for explanation of the table.

FIG. 14. Is a table showing human peptides matching collagen α1 (IX) inthe 30 kDa fragment. Database search result from the IonTrap analysis ofthe Asp-N digested 30 kDa band originating from the MMP-13 digestionmedium separated on α16% SDS PAGE

FIG. 15. Is a table showing the “include mass list”. Five peptide massesoriginating from the amino acid sequence of human collagen α1 (IX) NC4were chosen for MSMS analysis by the reversed phase LC ESI-Qtof MSsetup. The two first are the two most C-terminal peptides found in theAsp-N digest of the 30 kDa band of the MMP-13 digestion medium. Thethird and the fourth were easily detected by the reversed phase LCESI.IonTrap MS run in parallel and were included as controls. The fifthand last represents the C-terminal peptide expected if the cleavage sitewould have been the same as in bovine.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF

Initially bovine work is discussed and further down the details of theinvention in humans.

Interleukin-1 induced degradation of bovine nasal cartilage revealedrelease of fragments, such as the NC4 domain of the type IX collagen α1chain. The novel peptide antibody of the invention, against the epitopein the N-terminal part of the NC4 domain confirmed the finding.

Mass spectrometric analysis of the two most abundant fragments revealedthat the smallest fragment contained almost the entire NC4 domain,cleaved at a novel site between arginine-258 and isoleucine-259 in thesequence ETCNELPAR (SEQ ID NO:1) (-ETCNELPAR²⁵⁸—COOH) and -ITPGARSP (SEQID NO:2) (NH₂-ITPGARSP). A larger fragment contained the NC4 domain andthe COL3 domain with a novel cleavage site located between glycine-400and threonine-401 in COL3, RGPPGPPGPPGPSG- (SEQ ID NO: 3)(RGPP(OH)GP(OH)PGPP(OH)GPSG⁴⁰⁰—COOH) and NH₂-TIGFHDGD (SEQ ID NO:4),(NH₂-TIGFHDGD-) close to the NC3 domain.

Results of Bovine Study

Bovine Amino Acid Sequence

The amino acid sequence of bovine collagen α1 (IX) that matched to massspectrometric analysis in this study can be found in the non-redundant(nr) database from “National Center for Biotechnology Information” underthe name: gi|119901059 (22 Dec. 2006). Alignment of this sequence fromNCBI with the human α1 (IX) sequence (SwissProt P20849 reveals majordiscrepancies N- and C-terminally (Suppl. 1). The N-terminal part of thebovine sequence is 332 amino acids longer than the human sequence. Inthis study, no mass spectrometric data matched to any parts of this“additional” sequence. The bovine sequence shows 90% similarity to thefirst 642 amino acids of the human counterpart, while 279 amino acids inthe C-terminal are missing. This bovine sequence is likely to representan artifact to be replaced. We have therefore chosen to numberannotations in this text referring to the human sequence. Databasesearch results maintain their bovine sequence numbers.

Bovine Nasal Cartilage Explant Culture

Proteins released into the medium from IL-1 stimulated cartilageexplants (3, 6, 9, 12, and 16 days) were separated using 2D SDS PAGE andidentified through peptide mass fingerprinting. Gels from day 3 and day6 showed irregular separations particularly at the top part, typical ofsamples containing large amounts of intact and fragmented aggrecan thatis too large to enter the gel. In this project focus was on thesubsequent degradation after aggrecan release, but before the majorcollagen release (42). Collagen α1 (IX) was identified at differentpositions on 2D gels of mediums from day 12 and 16 (a typical gel shownin FIG. 1). The same spots were not present in the control samplesincubated without IL-1 (data not shown). A set of faster migratingprotein spots had an apparent mass of 28 kDa and the sequence coverageas found by peptide mass fingerprinting (13 peaks matched peptides, FIG.11 a) indicated that they represented a major portion of the NC4 domainof collagen α1 (IX). Peptide mass fingerprinting of the collagen α1 (IX)fragment migrating with an apparent mass of approximately 50 kDarevealed 23 peaks that matched peptides (FIG. 11 b), whereof ten werefrom the COL3 domain and the remaining from NC4. The peptide coverage ofthe slowest migrating spot identified as collagen α1 (IX) (apparent mass100 kDa) included in addition to NC4 and COL3, parts of the COL2 domain(25 peaks matched peptides, FIG. 11 c). It might represent crosslinkedparts or the full size collagen α1 (IX), and was not further studied(the bovine C-terminal sequence not known).

Peptide Antibody

In order to confirm and further enable the study of the identifiedcollagen α1 (IX) fragment, a peptide (NH₂-CGQDDLPGFDLISQFQ⁶⁴—CONH₂) (SEQID NO:14) from the N-terminal part of the α1 (IX) NC4 domain wassynthesized with an added cysteine coupled to Key limpet hemocyanin andused to immunize a rabbit. This NC4-antiserum showed binding to theNC4-peptide in solid state ELISA (titer 1:50000). The binding wasinhibited by addition of the NC4-peptide. A peptide concentration of 0.3ng/ml showed efficient inhibition of the binding.

Western Blot

Western blots using the polyclonal NC4-antiserum confirmed the presenceof collagen α1 (IX) fragments at 28 kDa and 50 kDa in medium from IL-1stimulated cartilage cultures separated on SDS PAGE. An additional bandat a position corresponding to 40 kDa indicated the presence of anothercollagen α1 (IX) entity initially released somewhat earlier than theother two (FIG. 2). These three fragments are referred to as the 28 kDa,40 kDa, and 50 kDa fragments respectively.

Identification of Cleavage Sites

The presence of collagen α1 (IX) fragments released into the medium frombovine nasal cartilage stimulated with IL-1 encouraged us to search forcleavage sites. 2D gel pieces containing the 28 kDa fragment weretreated with trypsin or AspN prior to LC MSMS (Qtof). Mascot databasesearches (Matrix Science Inc. Boston Mass., USA) using NCBInon-redundant (nr), carbamidomethyl (C), variable oxidation (M), andvariable hydroxylation (P, K) only identified peptides from the NC4domain. The most C-terminal peptide found in both the trypsin and AspNdigested samples was ETCNELPAR²⁵⁸ (SEQ ID NO:1) (FIG. 3). This wouldrepresent a peptide cleaved correctly at both its terminals by trypsinand a peptide cleaved correctly by AspN at its N-terminal whereas theC-terminal cleavage has to be mediated by another enzyme prior toprocessing. In FIGS. 2 a and 2 b peptides matched to MSMS data from theAspN digested sample is tabulated. A Mascot MSMS Ion search for peptidesin the AspN digest using trypsin specificity showed only the “cleavage”ETCNELPAR²⁵⁸ (SEQ ID NO:1) peptide, demonstrating that there had been notrypsin attack on the identified fragment. Tryptic digests of a 28 kDaspot contained, in addition to other NC4 peptides, three N-terminalpeptides, covering amino acids Phe³¹-Lys⁴⁶, Val³³-Lys⁴⁶, and Ser³⁵-Lys⁴⁶respectively (FIG. 12 c and FIG. 7). The two latter, both havenon-tryptic cleavages N-terminally, indicating that two new N-terminalsare present. Existence of different N-terminals have previously beenreported in NC4 isolated after collagenase digestion (43;44), but weobserved none of these cleavages in our set of data.

The 50 kDa collagen α1 (IX) fragment was digested in-gel with trypsinand analyzed by reversed phase ESI-IonTrap MS. Nine peptides weresequenced and matched to the NC4-COL3 domains of collagen α1 (IX),whereof the most C-terminal peptide) (RGPP*GPP*GPP*GPSG⁴⁰⁰) (SEQ IDNO:3) had a C-terminal cleavage not generated by the trypsin used (P*denotes hydroxyproline residue. Data not shown).

The minor 40 kDa fragment was identified as collagen α1 (IX) withsequence coverage in the NC4 domain only. It was not further studied.

MMP-13 Digestion of Bovine Nasal Cartilage

Digestion of bovine nasal cartilage with MMP-13 for 24 hours had avisual impact on the cartilage, similar to the IL-1 incubation, suchthat the tissue started to become transparent. Western blot of themedium using the NC4-antiserum indicated that two collagen α1 (IX)fragments containing the NC4 domain were present. Upon SDS-PAGE, theymigrated identically to the 28 kDa and 50 kDa fragments, as demonstratedby Western blot using the anti NC4 peptide-antiserum (FIG. 4 a). Uponlong exposure, also a weak 40 kDa band appeared, similar to the onefound in medium from IL-1 treated cartilage (FIG. 4 a inset). The gelpieces with the two bands at 28 kDa and 50 kDa from a Coomassie stainedgel were digested with AspN and trypsin, respectively. Analysis byreversed phase LC ESI-IonTrap MS identified N-terminal fragments ofcollagen α1 (IX). The most C-terminal peptide found in the 28 kDafragment was ETCNELPAR²⁵⁸ (SEQ ID NO:1), the same peptide as in IL-1stimulated bovine cartilage medium samples from day 12 and 16 (FIG. 3).The peptides identified (by reversed phase LC IonTrap MS) for the MMP-13generated 50 kDa collagen α1 (IX) fragment are listed in FIG. 13. Themost C-terminal peptide had a C-terminal cleavage not generated by thetrypsin (FIG. 4 b), indicating a newly identified cleavage site. Thesame peptide was detected in the sample of IL-1 treated cartilage (datanot shown), in addition to another COL 3-domain peptide (the same m/z,charge state, and retention time was found in the MMP-13 sample, but noMSMS was performed).

Discussion

This model of tissue breakdown in joint disease where cartilagedegradation is induced by IL-1 stimulation has been frequently used instudies of fragmentation and release of tissue macromolecules. It hasbeen shown that aggrecan fragments are initially released, followed byCOMP, Fibromodulin, and last collagen (45-47). In medium from cartilagecultured in the presence of IL-1 we found, using 2D gel electrophoresisfollowed by MALDI MS, release of collagen α1 (IX) fragments. The mostabundant fragment detected on 2D gels, with an apparent MW of 28 kDa,represented large parts of the globular NC4 domain. The trailing of the28 kDa fragment spots on 2D gel over a large interval of isoelectricpoints, might be due to deamidation of asparagine residues (48). Indeedone of the peptides sequenced appeared to exhibit deamidation (FIG. 7).This phenomenon was not further investigated. The larger 50 kDa fragmentwas shown to contain NC4 as well as large parts of the COL3 domain.

While type IX collagen domains COL 1, 2, NC 1, and 2 are tightlyassociated with the type II collagen fibril, COL 3 and the globular NC4protrude out from the fiber surface (49). This part of the type IXcollagen may serve a role as a connector between type II fibrils andother extracellular structural entities. In support, it was recentlyshown that the absence of type IX collagen could make the cartilage morevulnerable to an autoimmune attack by pathogenic antibodies (50) andreduce its ability to retain matrilin-3 (51). Fragmentation of type IXcollagen and loss of the functional NC4 domain from the tissue mightthus reflect an important and early step in the process of dismantlingcartilage.

The collagen α1 (IX) peptide antibody, as described above, was raisedagainst the synthetic peptide (C)GQDDLPGFDLISQFQ⁶⁴ (SEQ ID NO:14)representing a sequence close to the N-terminal of the NC4 domain. Thepeptide was chosen to be C-terminal of one previously used as theimmunogen for an antibody against the NC4 domain (52) since theseauthors described the existence of multiple N-terminals, where some werelocated in the immunogen sequence they used. Their antibody has beenused to describe changes of type IX collagen particularly in the growthplate (53-56). Our mass spectrometry data also demonstrated presence ofmultiple cleavages at the N-terminal of the NC4 domain (12c and FIG. 7),but none correspond to those previously described. It is not known ifthese cleavages are the results of IL-1 stimulation or if they arealready present in the tissue prior to stimulation.

Western blots with our new NC4-antiserum clearly indicated the presenceof three different type IX collagen fragments in medium from day 9, 12,and 16 (FIG. 2). The apparent masses of two of them match earlieridentified spots from 2D gels corresponding to approximately 28 kDa and50 kDa. The third fragment with an apparent mass of 40 kDa was observedalready in day 9 medium. Upon trypsin digestion and mass spectrometry ofthe 40 kDa fragment found in medium from IL-1 stimulated cartilage atday 12, the NC4 domain of collagen α1 (IX) was identified. The apparentmass suggests a fragment larger than the NC4 domain, but data from massspectrometry shows limited coverage and did not include any peptidesfrom the COL3 domain. The most abundant 28 kDa fragment was selected formore extensive characterization. The larger fragments were expected tobe more complex to study due to the collagenous domains and theirheterogeneity in hydroxylation of proline residues and the difficultyfor enzymes such as trypsin to cleave N-terminally of proline.

Digestion with trypsin and endoprotease AspN combined with reversedphase liquid chromatography on-line with electrospray mass spectrometry(LCMSMS) revealed a neocleavage in the C-terminal part of NC4, a fewamino acids N-terminal to the COL 3 domain. The most C-terminal fragmentfound with trypsin was ETCNELPAR²⁵⁸ (SEQ ID NO:1) (I). In order tosearch for peptides covering parts further to the C-terminal, the NC4containing fragment was cleaved with AspN. It produced the same mostC-terminal peptide ETCNELPAR²⁵⁸ as trypsin, despite that the enzyme willnot produce a cleavage between the PAR²⁵⁸ and the following isoleucine(FIG. 3). The AspN digest did not contain any other peptides that couldhave been generated by trypsin, excluding any contamination by thisenzyme. As another control, AspN digestion of the longer 50 kDa collagenα1 (IX) fragment did not produce an ETCNELPAR (SEQ ID NO:1) peptidedetectable with reversed phase LC ESI-IonTrap MS, whereas trypsin indeedcleaved at the arginine residues to produce this fragment (data notshown). Trypsin digestion of the 50 kDa collagen α1 (IX) fragment,subsequently analyzed by reversed phase LC ESI-IonTrapMS identified twopeptides in the COL3 domain in medium from IL-1 day 16. The mostC-terminal of them has a C-terminal non-tryptic cleavage(-RGPPGPPGPPGPSG⁴⁰⁰—COOH (SEQ ID NO:3), NH₂-TIGFHDGD-) (SEQ ID NO:4),suggesting that this cleavage had occurred in the tissue during IL-1stimulation (FIG. 13 and FIG. 4 b). The 28 kDa and the 50 kDa fragmentsare released simultaneously from the tissue (FIG. 2) and are thus likelycleaved in parallel, with formation of the 40 kDa fragment preceding.

In previous experiments (57) it was shown that MMP-13 appeared in anactive form and cleaved fibromodulin starting around day 9 whencartilage was treated with IL-1. Although the cleavage site infibromodulin (between -PAY⁶³ A⁶⁴YG-) differs from the one found in typeIX collagen (-PAR²⁵⁸ I²⁵⁹ TP-), we decided to test whether digestion ofcartilage with MMP-13 could generate the cleavage of collagen α1 (IX)NC4 that we found in medium from IL-1 stimulated cartilage explants.

Bovine nasal cartilage explants were digested with MMP-13 and indeed theenzyme cleaved collagen α1 (IX). The appearance of NC4 containingfragments in the medium as shown by Western blot using the NC4-antiserumwas very similar to those from IL-1 induced cleavage (FIG. 4 a). The twofragments, 28 kDa and 50 kDa from collagen α1 (IX) NC4 previouslyobserved upon IL-1 stimulation, were released from the bovine cartilageinto the medium upon MMP-13 digestion. The 40 kDa band that was seen inIL-1 treated cartilage at day 9, 12, and 16, could be seen as a faintband in this blot upon longer exposure times (FIG. 4 a inset). The 28kDa fragment in medium from MMP-13 digested bovine cartilage wassubsequently identified as the NC4 domain of collagen α1 (IX) byreversed phase LC ESI-IonTrap MS. It contained the same most C-terminalpeptide (ETCNELPAR²⁵⁸) (SEQ ID NO:1), as previously found in the mediumfrom IL-1 stimulated bovine cartilage.

The 50 kDa fragment observed in media from both IL-1 stimulated bovinecartilage from day 16 and MMP-13 digested bovine cartilage were shown tocontain peptides from the NC4 as well as the COL3 domains. Preliminaryanalyses of the 50 kDa fragment released both upon IL-1 and MMP-13treatment of cartilage revealed in both cases the same C-terminalcleavage peptide (R³⁸⁷ GPP*GPP*GPP*GPSG⁴⁰⁰—COOH (SEQ ID NO:3) with threehydroxylated* residues) generated, in the C-terminal parts of the COL3domain, prior to our analyses. It is probable that MMP-13 being acollagenase induced this cleavage also in the IL-1 treated sample. It islikely that the enzyme causing the cleavage in the IL-1 stimulatedcartilage indeed is MMP-13, since one substrate site defined representsone for a collagenase and a second is for a different enzyme, such as agelatinase. MMP-13 represents an enzyme known to have these twoactivities.

Removal of type IX collagen as a step in tissue maturation has beendiscussed in earlier studies (58;59). The latter reported that the NC4domain was removed at the initiation of mineralization, and as themineralization progressed both type II and IX collagen were removed fromthe matrix. The cleavage was not identified and it is not possible toconclude whether it was the same as any of those observed here. Thepresence of MMP-13 in hypertrophic chondrocytes has previously beenobserved using in situ hybridization (60;61). Combined with the resultsreported in our present study, it seems plausible that collagen IX isremoved from growth plate fibrils by MMP-13, as a step in endochondralbone formation.

The presented data elucidates the degradation of an important cartilagecomponent in a model system and pinpoints MMP-13 as a protease with theability to accomplish specific cleavages in collagen IX (FIG. 9, 10).Sites of non-triple helix as well as such for triple helical parts werefound. The new sites disclosed in the invention, can be used to developantibodies only recognizing cleaved molecules and could be used in thedevelopment of new diagnostics procedures by molecular markertechnology.

Results of Human Study

Digestion of Human Articular Cartilage with MMP-13 and Determination ofNovel Cleavage Site

Human articular cartilage showed no visible signs of degradation uponMMP-13 digestion, in contrast to bovine nasal cartilage. Even so, as thedigestion medium was analyzed with Western blots (using the NC4antiserum) it showed reactivity (FIG. 5 a). The band had an apparentmass of 30 kD, which distinguished it from the previously describedbovine fragment. A band corresponding to that stained in Western blotwas cut out from Coomassie stained gels, digested with Asp-N or Glu-C,and analyzed with reversed phase LC ESI-IonTrap MS. In both digests typeIX collagen α1 NC4 was identified (Asp-N result in FIG. 11), as well asa peptide representing a potentially new cleavage siteCHELPARITPSQ²⁶³-COOH (SEQ ID NO:6) NH₂-TTDERGPP- (SEQ ID NO:7). The newcleavage site is five amino acids C-terminal of the bovine cleavagesite, -ETCNELPAR²⁵⁸-COOH (SEQ ID NO:1) NH₂-ITPGARSP- (SEQ ID NO:2) foundin the 28 kD fragment. No masses matching a fragment cleaved at thebovine cleavage site were found in the human sample.

In order to improve and confirm the sequence data of the potentialcleavage site peptide, the ESI-Qtof mass spectrometer was used. In ourhands the Qtof system is 5× less sensitive compared to the IonTrap, thusthree gel bands were pooled and digested with endoproteinase Asp-N inammonium bicarbonate buffer to yield cleavages N-terminal of asparticacid (D) and glutamic acid (E).

To avoid that the instrument was occupied running MSMS on an arbitrarypeptide eluting prior or at the same time as the potential cleavage sitepeptide the instrument was set to identify and perform MSMS sequencingon a limited number of peptide masses (FIG. 12). The following peptideswere chosen: two internal control NC4 peptides known to elute late, twopotential novel cleavage site peptides with zero or one missed Asp-Ncleavage, and the human peptide equivalent to the previously identifiedcleavage site peptide in bovine cartilage. The control peptides and thetwo potentially novel cleavage site peptides had been detected usingESI-IonTrap mass spectrometry, and we now set out to use ESI-Qtof massspectrometry to confirm the MSMS data of the two latter peptides. In theQtof experiment both control peptides and the two peptides originatingfrom the new human cleavage site were sequenced and found to match thetype IX sequences (MSMS data from one peptide shown in FIG. 5 b). Thehuman peptide equivalent to the bovine cleavage site peptide was notpresent in the Qtof analyses (nor in IonTrap analysis).

Neoepitope Peptide Antiserum (CPA-antiserum)

Western blots of the medium from the human cartilage digested withMMP-13 was performed by separating the digest on 16% SDS PAGE andidentification using the neoepitope peptide antiserum. A band at thesame position as that observed with the antibody to NC4 was identified(FIG. 6). Synovial fluid from an osteoarthritis patient (in contrast toan rheumatoid arthritis patient) showed strong inhibition in anInhibition ELISA using the affinity purified CPA-antiserum (FIG. 8). Theexperiment also showed that the addition of SDS was essential for thesynovial fluid epitope to be recognized by the CPA-antiserum.

EXAMPLE 1 Bovine, Collagen α1 (IX) NC4-Peptide Antiserum

A 15-mer peptide sequence of the N-terminal portion of the type IXcollagen α1 NC4 domain showing potential as an immunogen and no homologyto other sequences found in a BLAST search was selected forimmunization. The sequence is identical in human, mouse, rat, cow,chicken, dog, chimpanzee, and rhesus monkey and was represented inmedium from IL-1 stimulated cartilage. An N-terminal cysteine was addedfor coupling, giving the sequence for immunizationNH₂-CGQDDLPGFDLISQFQ⁶⁴—CONH₂ (SEQ ID NO:14). The peptide coupled toKeyhole Limpet Hemocyanin (KLH) was used for immunization of a rabbit.Peptide synthesis and antibody production were custom services providedby Innovagen AB (Lund, Sweden). The peptide and the antiserum will bereferred to as NC4-peptide and NC4-antiserum respectively.

EXAMPLE 2 Neoepitope Peptide Antiserum (Human)

The peptide (C)-PARITPSQ²⁶³—COOH (SEQ ID NO:13), representing theC-terminal part of the released al (IX) fragment, was synthesized,coupled to Keyhole Limpet Hemocyanin (KLH), and used to immunize arabbit. The antiserum was affinity purified using a column with peptidecoupled to agarose. Peptide synthesis, peptide column production andantibody production were custom services provided by Innovagen AB (Lund,Sweden).

EXAMPLE 3 Western Blot (Bovine)—FIGS. 2,4 and 5 a

Samples of medium from bovine cartilage digested with MMP-13 wereseparated by 16% polyacrylamide-SDS gel electrophoresis and thentransferred to a nitrocellulose membrane (Hybond-C, AmershamBiosciences) (38). Blocking was performed overnight with 3% (w/v)low-fat dry milk in Tris-buffered saline (TBS), pH 7.4 and 0.2% (v/v)TWEEN 20™ at 4° C. The membrane was rinsed with the blocking solution(described above) omitting milk and incubated at room temperature withthe NC4-antisera diluted 1:1000 in 2% (w/v) milk, TBS, and 0.2% TWEEN 20for 1 hour. After rinsing, the nitrocellulose membrane was incubated for1 hour at room temperature with a peroxidase-conjugated secondaryantibody diluted 1:30000 (AffiniPure donkey anti-rabbit IgG JacksonImmunoResearch) in 2% milk/TBS/TWEEN 20. Blots were activated for 1minute using home made ECL-reagents (20 ml 0.1 M Tris-HCl pH 8.5, 48 μl250 mM Luminol in DMSO, 48 μl 40 mM p-coumaric acid in DMSO, and 14 μlH₂O₂). Agfa CRONEX 5 medical X-ray films were exposed to the membranesfor an appropriate time period and automatically developed with an AgfaCurix 60.

EXAMPLE 4 Western Blot (Human)

The affinity purified neoepitope antiserum used in Western blotting ofmedium from human cartilage digested with MMP-13 was diluted 1:100,while all other conditions are described in example 4.

EXAMPLE 5 Inhibition ELISA

A Nunc Maxisorp (no 446612) was coated with 0.1 μg/mlGGGPARITPSQ²⁶³—COOH (SEQ ID NO:15) peptide in PBS at room temperatureover night in a humid box. Ovalbumin, 2 mg/ml, in PBS was used to blockfurther binding. The peptide standard (0-10 μg/ml) or synovial fluidfrom osteoarthritis (1:10, 1:40, and 1:160) was dissolved in 0.8% SDS,0.5% ovalbumin, and 1% pig serum and preincubated on a sterilin plateover night at room temperature in a humid box. Each peptideconcentration and synovial fluid dilution was repeated in triplicates.The affinity purified neoepitope antiserum was diluted 1:1000 in 4%triton, 1% pig serum and mixed with the preincubated peptide standardand synovial fluids, prior to transfer to the Nunc plate. Secondaryantibody was pig anti rabbit immunoglobulins coupled to alkalinephosphatase (Dako-306) at 1:1000 in 1% pig serum. Paradinitrophenylphosphate at 1 mg/ml was used as substrate (9.7% diethanolamine, 0.01%MgCl₂×6H₂O, 0.02% NaN₃). The absorbance reading at 405 nm was doneimmediately after substrate addition and then after one hour incubation.The first reading (zero reading) was subtracted from the second reading,the results from three wells treated similarly were averaged, andplotted against peptide concentration or sample dilution.

EXAMPLE 6 Study Comparing the Absence/Presence of NC4 with Bone MineralDensity

5 postmenopausal women with a distal radius fracture, are studied. Thepatients are treated with a plaster splint for 4 weeks. The bone mineraldensity (BMD) of the forearm bones is measured with dual-energy x-rayabsorptiometry (DEXA) 2, 4, 6 and 8 weeks after the fracture. At thesame occasions serum samples are taken. The results of BMD are comparedto the presence or absence of NC4 in the serum samples. The detection ofNC4 is done according to example 5.

The bone mineral density at fracture site was 28%, 46%, 86%, 103% of thevalue obtained of the control limb 2, 4, 6 and 8 weeks after thefracture. In all the 5 cases the change of bone mineral density at thefracture site exhibited a correlation with the presence/absence of NC4.At the 2, 4, 6 weeks occasions NC4 is detected while it is absent at the8 week occasion. The presence/absence of NC4 can accurately determinewhen the fracture is fully repaired. In conclusion, the amount of NC4can be used as a new technique in monitoring fracture healing.

Therefore, the foregoing is considered as illustrative only of theprinciples of the invention. Further, since numerous modifications andchanges will readily occur to those skilled in the art, it is notdesired to limit the invention to the exact construction and operationshown and described, and accordingly, all suitable modifications andequivalents, including functional unimportant variations of sequences,may be resorted to, falling within the scope of the invention, and minorvariations are deemed to be equivalent to the particular sequencesherein set forth.

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Rheumatoid        Arthritis New Frontiers in Pathogenesis and Treatment, Oxford        University Press, Oxford

What is claimed is:
 1. An isolated antibody, comprising an antibodyhaving specific reactivity with a cleavage neoepitope of collagen IX,the neoepitope selected from the group consisting of PARITPSQ (SEQ IDNO: 13), TTDERGPP (SEQ ID NO 7), RGPPGPPGPPGPRG (SEQ ID NO: 9) andTIGFHDGD (SEQ ID NO: 10), wherein the isolated antibody does not reactwith full length collagen IX and is useful for detection of collagen IXcleavage.
 2. A diagnostic kit useful in detecting or monitoring a humandisorder affecting connective tissues, comprising an antibody havingspecific reactivity with a cleavage neoepitope of collagen IX, theneoepitope selected from the group consisting of PARITPSQ (SEQ ID NO:13), TTDERGPP (SEQ ID NO 7), RGPPGPPGPPGPRG (SEQ ID NO: 9) and TIGFHDGD(SEQ ID NO: 10)), wherein the isolated antibody does not react with fulllength collagen IX and the diagnostic kit is useful for detection ofcollagen IX cleavage.